Freezing adversely affects measurement of vascular endothelial growth factor levels in human aqueous samples

Sankarathi Balaiya Sandeep Grover Ravi K Murthy Kakarla V ChalamDepartment of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USAPurpose: Aqueous levels of vascular endothelial growth factor (VEGF) can be a surrogate marker of intraocular VEGF activity and a measure of efficacy of anti-VEGF treatment in a variety of vasoproliferative retinal disorders, including diabetic retinopathy, age-related macular degeneration, and central retinal vein occlusion. Measurement of the VEGF level may be adversely affected by premeasurement variables, such as freezing and delay, in sample analysis. We aim to evaluate the effect of storage and delayed measurement of human aqueous VEGF levels in these conditions.Methods: Aqueous samples collected from patients receiving intravitreal injection of bevacizumab for various retinal diseases were divided into two groups. In Group 1, the VEGF levels were analyzed on the same day; in Group 2, the VEGF levels were analyzed after 21 days of freezer storage (-80°C) using immunobead assay. Statistical comparison using a paired t-test was performed between the two groups.Results: Thirty-one aqueous humor samples were collected, and the VEGF concentration for fresh samples was 7.8 ± 5.9 pg/mL (mean ± SD) compared to 6.5 ± 6.0 pg/mL in frozen samples, resulting in a statistically significant difference (P = 0.03).Conclusions: Accurate measurement of the VEGF level is a vital component of clinical decision-making. Delayed analysis of VEGF levels in aqueous samples may result in significant sample degradation and lower levels of measured VEGF.Keywords: VEGF level, aqueous humor, immunobead assay, VEGF storag

Effects of Indocyanine green on cultured retinal ganglion cells in-vitro

<p>Abstract</p> <p>Background</p> <p>Indocyanine green (ICG) dye is commonly used to stain the inner limiting membrane during macular surgery. There are reports documenting the toxicity of ICG on retinal pigment epithelial cells, with conflicting results in retinal ganglion cells. In the present study, we evaluated the effect of ICG on retinal ganglion cells in vitro.</p> <p>Cultured rat retinal ganglion cells (RGC-5) were exposed to different concentrations of ICG (0.25, 0.5, 1.0, 1.25, & 5 mg/ml) and at various time intervals (1, 5, 15, 30, & 60 minutes). Changes in structural morphology were identified using phase contrast bright field microscopy. Cell viability was quantified using the neutral red assay and cell death was characterized using Annexin-V staining.</p> <p>Findings</p> <p>Significant morphologic changes were observed at the 15 and 60 min intervals for all concentrations, where a reduction in cell size and loss of normal spindle shape was noted. A dose dependent decrease in cell viability was observed with increasing concentration of ICG as well as increasing exposure intervals. Compared to control, 48-74% reduction in neutral red uptake at all concentrations for exposures 5 min or greater (p < 0.001). Even at 1 min exposure, a dose dependent decline was observed in cell viability, with a 28-48% decline for doses above 1.25 mg/ml (p = 0.007). Staining with Annexin-V, demonstrated a similar dose and time dependent increase in number of cells exhibiting early apoptosis. A greater than two-fold increase in Annexin-V expression for all doses at exposures greater than 1 min was noted.</p> <p>Conclusion</p> <p>ICG dye exhibits toxicity to retinal ganglion cells at clinically relevant doses following 1 min exposure.</p

Osmolarity and spectrophotometric property of brilliant blue green define the degree of toxicity on retinal pigment epithelial cells exposed to surgical endoilluminator

Sankarathi Balaiya, Kumar Sambhav, William B Cook, Kakarla V Chalam Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USA Objective: To evaluate the effect of varying concentrations of brilliant blue green (BBG) and their different biochemical characteristics on retinal pigment epithelial (RPE) cells under xenon light source illumination at varying distances to identify safe parameters for intraoperative use. Methods: Human retinal RPE cells (ARPE-19) were exposed to two concentrations (0.25 and 0.50 mg/mL) of BBG and illuminated with a xenon surgical illuminator at varying distances (10&nbsp;and 25 mm), intensity levels, and time intervals (1, 5, and 15 minutes). Additionally, the effect of osmolarity was examined by diluting BBG in different concentrations of glucose. Cytotoxicity of BBG and osmolarity effects on cell viability were evaluated using a WST-1 assay. Light absorption and emission characteristic of BBG in different solvents were measured using a plate reader at different wavelengths. Lastly, the activity of caspase-3 was also studied. Results: Cell viability of ARPE-19 cells was 77.4%&plusmn;12.7%, 78.7%&plusmn;17.0%, and 65.0%&plusmn;19.7% at 1, 5, and 15 minutes to exposure of high illumination xenon light at 10 mm (P&lt;0.05) compared to controls. At both distances of illumination (10 and 25 mm), similar cell viabilities were seen between 1 and 5 minutes of exposure. However, there was a decline in viability when the illumination was carried out to 15 minutes in all groups (P&lt;0.05). There was no significant reduction in cell viability in presence or absence of xenon light in different osmolar solutions concentrations of glucose (P&gt;0.05). Maximal light absorption of BBG was noted between 540 and 680 nm. Activated caspase-3 level was not significant in both the concentrations of BBG (P&gt;0.05). Conclusion: Our findings suggest that BBG at 0.25 mg/mL during vitreoretinal surgery is safe and not toxic to RPE cells up to 5 minutes under focal high illumination (10 mm) and up to 15&nbsp;minutes under medium diffuse illumination (25 mm). BBG was safe to be mixed with isotonic glucose solution at the concentration range of 2.5%&ndash;10%, regardless of the illumination status. Keywords: brilliant blue green dye, endoilluminator, vitrectomy, human retinal RPE cells, internal limiting membrane pee

Evaluation of ultraviolet light toxicity on cultured retinal pigment epithelial and retinal ganglion cells

Sankarathi Balaiya, Ravi K Murthy, Vikram&amp;nbsp;S Brar, Kakarla V ChalamDepartment of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USAPurpose: Our study is aimed at evaluating the role of UVB light in inducing cytotoxicity in an in vitro model.Methods: RGC-5 and ARPE-19 cells were exposed to different time periods of UVB light: 0, 15, 30, and 45 min. They were subsequently examined for changes in cell morphology, cell viability (neutral red uptake assay), generation of reactive oxygen species (ROS), expression of bax, bcl-2 and cytochome C by reverse transcriptase polymerase chain reaction and western blot, respectively.Results: Dose-dependent reduction in cell viability to UVB light was demonstrated with parallel increase in ROS. Increased duration of exposure (&amp;gt;15 minutes), was associated with increased expression of bax and cytochrome C, and absence of bcl-2 expression.Conclusion: UVB light exposure results in cell cytotoxicity. The concomitant generation of ROS and expression of apoptotic markers suggests the role of oxidative stress in UVB-mediated apoptosis in an in vitro model of retinal ganglion and pigment epithelial cells.Keywords: ultraviolet light, retinal pigment epithelium, retinal ganglion cell, reactive oxygen species, cytochrome

Modified protocol for <it>in vivo</it> imaging of wild-type mouse retina with customized miniature spectral domain optical coherence tomography (SD-OCT) device

<p>Abstract</p> <p>This protocol outlines and evaluates a modified scanning procedure for a customized spectral domain optical coherence tomography (SD-OCT) imaging apparatus within the wild-type C57Bl/6 mouse posterior segment. This modified protocol allows for the capture of a 50 degree field of view spanning 3 mm by 3 mm perimeter with the optic disc as the central point. By utilizing this scanning protocol a more reliable measurement of retinal thickness can be achieved outside the fluctuating region of the optic disc. This protocol, when applied to this high resolution device, enables non-invasive <it>in vivo</it> histological imaging and biometric assessment of the various layers of the rodent posterior segment within a 20 – 30 min procedural time-frame. This protocol could establish a standardized method for evaluating morphological changes, with this commercial SDOCT device, when assessing longitudinal disease pathophysiology and treatment response in mouse models for future vision science research.</p